The proposed research has two major objectives. The first is examination of those aspects of the solution structure of cytochrome c that influence the reactivity of the tyrosyl phenolic groups. This part of the work employs O-(1-13C)acetyl substituents of the phenolic groups as NMR-detectable probes of their surroundings. The tyrosyl residues of guanidinated horse heart cytochrome c have been specifically acetylated by reaction with N-(1-13C)acetylimidazole. 13C-NMR spectra of selected derivatives were obtained at pH 5-8, in the presence of cyanide ion, in the ferrous and ferric redox states, and after denaturation with 6 M quanidine hydrochloride. The O-(1-13C)acetyl tyr resonances give chemical shift values ranging from 171.8 to 176.0 ppm. These resonances were assigned to specific O-(1-13C)acetyl tyr groups based on the known order of tyr reactivity toward N-acetylimidazole. The chemical shift of O-(1-13C)acetyl tyr 67 was found to be particularly sensitive to changes in protein structure. Acetylation of tyr 74 gives rise to a pH-dependent equilibrium between conformers in the ferric state and a conformation change in the ferrous state. Activity measurements in the rat-liver succinate oxidase system indicate that these O-acetyl tyrosyl derivatives of cytochrome c are essentially identical or only slightly altered in activity relative to the native protein. The second objective is to study the interaction of cytochrome c with its mitochondrial site of action and related model systems, using these O-(1-13C)acetyl derivatives as NMR-detectable reporter groups.